Project Summary Ubiquitylation describes the process by which the small protein ubiquitin is covalently attached to other proteins. Most, if not all, cellular processes are regulated in some way by ubiquitylation. Over the past decade we have gained tremendous insight into the molecular components and processes that conjugate ubiquitin to substrate proteins. We also made significant progress in understanding how ubiquitylated proteins are targeted to the 26S proteasome for degradation. Indeed, most of our knowledge about ubiquitylation stems from studies of protein degradation. Protein ubiquitylation has become a synonym for protein degradation, and most of the current research is focused on the role of ubiquitin in targeting proteins for degradation by the 26S proteasome or the lysosomal cycle. However, comparative ubiquitin profiling experiments using proteasome inhibition estimate that only about 60% of ubiquitylated proteins are efficiently degraded by the proteasome, implying that protein ubiquitylation has widespread signaling functions outside the proteasome pathway. The type of ubiquitin modification, mono, multi, or poly-ubiquitylation, and ubiquitin chain topologies are involved in signal specification, but beyond that the understanding of molecular concepts governing ubiquitin signaling is rudimentary. Many examples in ubiquitin biology illustrate our lack of molecular understanding of signaling by ubiquitylation. Such detailed insight into processing of the diverse ubiquitin signals, especially proteolysis-independent ubiquitylation pathways, will be important for basic biomedical research and development of therapeutics targeting the ubiquitin system. Key questions are: Why are some ubiquitylated proteins degraded and others are not? How can ubiquitylation directly affect protein activity? What are the mechanisms of direct protein regulation by ubiquitylation and what are the components mediating regulation? Over the past years we have developed a highly defined system that allows us to study these questions in great detail. This system is focused on the cullin-RING ubiquitin ligase complex SCFMet30, which connects metabolic stress to cell cycle regulation. SCFMet30 modifies a number of substrates with the canonical degradation signal, the lysine-48 (K48) linked ubiquitin chain. Interestingly, while some substrates behave as expected and are targeted for degradation by the 26S proteasome, other substrates are regulated in a proteolysis-independent manner. This proposal builds on a plethora of tools available to analyze biochemistry and physiology of ubiquitin signaling in this system and will address: (1) how phosphorylation of a ubiquitin binding domain can dictate signal identity of the K48 ubiquitin chain in a spatially defined manner; (2) how a K48-linked polyubiquitin chain can directly regulate transactivation in a proteolysis-independent manner; (3) what components are necessary to recognize a K48-linked chain as a signal for active disassembly/remodeling of multisubunit protein complexes; (4) how proteasome substrates are selectively recognized and targeted to the proteasome; and (5) what are the concepts of ubiquitin signal recognition and its regulation at the atomic level. Ubiquitylation affects many important cellular processes and has been linked to a number of human diseases including cancer, neurodegeneration, and retroviral infection. A contribution of proteolysis- dependent and independent mechanisms is evident. It will be important to understand the molecular concepts that govern ubiquitin signaling to design diagnostic tools and treatment strategies. This proposal aims to achieve detailed mechanistic insight into signaling through ubiquitin and to define the concepts of proteolytic as well as regulatory ubiquitylation pathways.